Na*/H* exchangers (NHE) are integral plasma membrane proteins that catalyze the electroneutral exchange of extracellular Na* for intracellular H* in a one to one stoichlometry. The activity of this protein is important in the maintenance of a variety of cellular homeostatic processes; Including regulation of cellular pH, volume, and control of transepithellal absorption of electrolytes. In addition, the exchanger facilitates cellular growth and proliferation in response to various mitogens and growth factors. We report here the cloning, primary structure, heterologous expression, tissue distribution and localization of a cDNA encoding rat NHE5, a fifth member of the mammalian plasma membrane Na*/H* exchanger (NHE) gene family. The full-length open reading frame as well as 34 nucleotides of 5' and 1443 nucleotides of 3' untranslated sequences were obtained using a PCR strategy involving reverse transcription polymerase chain reaction (RT-PCR) and 5'/3' rapid amplification of cDNA ends (RACE). The NHE5 cDNA encodes a protein of 898 amino acids with a calculated Mr of 99,044 and is predicted to contain 11-13 transmembrane domains. An amino acid comparison of the coding region of rat NHE5 reveals 95% identity with human NHE5. Northern hybridization analysis showed that high level expression of NHE5 mRNA is restricted to brain. Transfection of the coding region of rat NHE5 into NHE-deficient PS120 cells resulted in Na*/H* exchange activity that was relatively insensitive to the amiloride analogue, 5-(N-ethyl-JV-isopropyl) amiloride, with a half-maximal Inhibitory concentration (Icg50) of 1.52 ( 0.25 (M. In situ hybridization of rat brain sections revealed significant NHE5 mRNA levels in the dentate gyrus with lower levels observed in the hippocampus and cerebral cortex. These results suggest a specialized role for this fifth NHE isoform in neuronal tissues. Key words: Na*/H* exchangers, rat NHE5, RT-PCR, cloning, functional expression